(B-64) Shear induced swelling and post-recovery mechanics of red blood cells

Introduction:: The basic function of a red blood cell (RBC) is to mediate the transport of oxygen from the lungs to the tissues. RBCs maintain a strict surface area to volume ratio (SVR) which accounts for their unique discoid shape and enables their passage through narrow blood vessels. Furthermore, it maximises the contact area between the RBC and neighbouring endothelial cells for the efficient transfer of gases. Deviations from the optimal SVR manifests in RBC swelling or dehydration, both of which compromise the biophysical properties of RBCs and impairs oxygen delivery.

RBCs circulate around the body and are exposed to shear stresses (SS) as high as 15Pa. SS induces mechanical deformation on the cell which activates mechanosensitive channels such as Piezo1 and the recently discovered TRPV2. The opening of these channels increases the permeability of the RBC membrane to cations such as Na⁺, K⁺ and Ca²⁺ which results in perturbations of the intracellular cation composition. As cellular volume is mainly regulated by movement of monovalent cations, this suggests that the RBC volume varies constantly with SS exposure.

Calcium (Ca²⁺) and Na⁺ K⁺ ATPase (ATP1) have been elucidated to play a critical role in maintenance of RBC volume. In this study, we demonstrate the dependence of RBCs on these pathways for effective RBC volume regulation.


Materials and Methods:: Washed, isolated RBCs were obtained from healthy male volunteers. RBCs were suspended in 1 x phosphate buffered saline (PBS) at 1% hematocrit. Uniform shear stress (SS) was maintained by using a cone-and-plate rheometer (DV2+, Brookfield, Canada). RBCs were sheared with a SS of 15 Pa for a total duration of 300s to inflict supraphysiological SS over a prolonged period to invoke the largest possible shear-induced size. Sheared RBCs were observed on a glass slide using an inverted microscope (IX-71, Olympus). RBC images were taken using a high-speed camera (FASTCAM-1024 PCI, Photron, USA). At least 300 individual RBCs were imaged in each sample for statistical comparison. Identification of the cell boundary and measurement of the 2D projected RBC area were achieved using an in-house developed MATLAB code. Temperature was set at 37°C to mimic the physiological state of blood circulation.

Two conditions were tested – (1) RBCs were sheared under physiological Ca²⁺ level (2mM) to evaluate the contribution of Ca²⁺ to volume regulation and compared against RBCs sheared in media devoid of Ca²⁺; (2) ATP-reduced RBCs generated by incubation in 37°C water bath for 8 hours were sheared in Ca²⁺-free media to assess the energy-dependent non-Ca²⁺ pathways of RBC volume regulation. Longitudinal assessment across 40 minutes was performed to determine the recovery of the swelled RBCs in both fresh and ATP-reduced conditions.

Medium Ca²⁺ and K⁺ levels were measured using blood analysis system (epoc, Siemens, DE). ATP levels were measured using luminescent ATP detection assay kit (A22066, Thermo Fisher Scientific, USA).


Results, Conclusions, and Discussions:: In the presence of Ca²⁺, there was no significant change in RBC size after shearing (0 ± 0.210%). However, in the absence of Ca²⁺, there was a significant increase in RBC size after shear treatment (3.48 ± 0.0642%) (Figure 1A). Comparing medium K⁺ levels, both groups showed an increase in K⁺ levels with RBCs sheared in Ca²⁺-free media showing a lower increase in K⁺ release (with Ca²⁺: 22.8 ± 2.61%, without Ca²⁺:13.9 ± 2.51%) (Figure 1B).

ATP measurements revealed a reduction in intracellular ATP levels by 50.9% in the ATP-reduced RBC samples (Figure 1C). Conversely, there was no significant change in RBC deformability in the ATP-reduced RBC samples (Fresh: 0.508 ± 0.00177 vs ATP-reduced: 0.509 ± 0.00274) (Figure 1D). Fresh RBC samples showed an exponential size recovery after shearing (R²: 0.974), albeit unable to return to their original size. ATP-reduced RBC samples showed a similar behaviour (R²: 0.975) but with a reduced recovery rate (fresh: 3.93 ± 1.10 minutes vs ATP-reduced: 20.2 ± 5.40 minutes) and a reduced amount (Fresh: 55.9 ± 3.22% vs ATP-reduced: 43.0 ± 6.10%) (Figure 1D).


The insignificant size change for RBCs sheared in Ca²⁺ media affirms the involvement of Gardos effect in preventing cell swelling through K⁺ efflux. Furthermore, reduced K⁺ efflux in RBCs sheared in Ca²⁺-free media supports the theory of volume fluctuation caused by K⁺ transport and highlights the involvement of a separate regulatory mechanism (i.e. ATP1).

A decrease in ATP results in reduced cellular activity, in particular, ATP1 which has bearing on cell volume regulation. Insignificant differences in cell deformability implies that the generated ATP-reduced RBCs do not present marked biophysical changes. In the absence of Ca²⁺, while RBCs showed signs of size recovery with time, the rate of recovery was in the order of minutes and the RBCs were unable to recover back to its original size. This behaviour was observed to be energy dependent, evident from the reduced rate and extent of recovery in ATP-reduced RBCs. Our study shows the critical role Ca²⁺ plays in volume regulation and its significance in allowing RBCs to perform its basic function effectively.


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